Among plant transgene expression systems, expression of a transgene under the control of a heterologous promoter has been in use for several years. Apart from such conventional plant expression systems, virus-based expression systems can be used for rapid protein production in plants (for review see: Porta & Lomonossoff, 1996, Mol. Biotechnol., 5, 209-221; Yusibov et al., 1999, Curr. Top. Microbiol. Immunol., 240, 81-94) and are a powerful tool for functional genomics studies (Dalmay et al., 2000, Plant Cell, 12, 369-379; Ratcliff et al., 2001, Plant J., 25, 237-245; Escobar et al., 2003, Plant Cell, 15, 1507-1523). Numerous publications and patents in the field describe systems based on DNA and RNA viral vectors (Kumagai et al., 1994, Proc. Natl. Acad. Sci. USA, 90, 427-430; Mallory et al., 2002, Nature Biotechnol. 20, 622-625; Mor et al., 2003, Biotechnol. Bioeng., 81; 430-437; U.S. Pat. Nos. 5,316,931; 5,589,367; 5,866,785; 5,491,076; 5,977,438; 5,981,236; WO02088369; WO02097080; WO9854342). The existing viral vector systems are usually restricted to a narrow host range in terms of their best performance and even the expression level of such vectors in their most favourable host is far below the upper biological limits of the system. An important issue of virus-based systems is the method of delivery of the viral replicon to a plant cell. The most broadly applied method of delivery for large-scale production (simultaneous production in many plants, e.g. in a farm field or a greenhouse) is the use of infectious copies of RNA viral vectors (Kumagai et al., 1995, Proc. Natl. Acad. Sci. USA, 92, 1679-1683). Because of a relatively high tendency of recombinant viral RNA vectors to lose the heterologous inserts during the cycles of their replication, the method requires transcription of DNA templates in vitro, and as a result is inefficient and expensive. Another approach to solve the delivery problem could be the presence of a viral RNA replicon precursor in each cell of a transgenic plant, such that it can be released upon triggering the replication process by complementing a function of the viral vector (e.g. using helper virus—U.S. Pat. No. 5,965,794) or using other regulated switch systems (e.g. site-specific recombination—U.S. Pat. No. 6,632,980).
Despite many publications in the field including patented technologies, there are still no large scale virus-based production systems that work with sufficient efficiency and yield for commercial high-yield production, predominantly due to two main reasons:
Firstly, transient plant virus-based expression systems are generally restricted to specific hosts, which may not be suitable for large scale cultivation due to their susceptibility to environmental factors. Moreover, they are generally restricted to certain parts of a plant host, thus excluding most of the plant biomass from the production process and as a result minimizes the relative yield of the recombinant product per unit of plant biomass down to a level comparable to that achievable by a conventional transcription promoter in a transgenic plant;Secondly, attempts to scale up the virus-based production system by generating transgenic plant hosts having the viral replicon precursor stably integrated in each cell have not provided a solution either, in particular because of underperformance of said replicons in such position, “leakiness” of the gene of interest to be expressed from said replicon and lack of an efficient switch system for said vectors. Certain progress was achieved with PVX-based vectors by using suppressors of PTGS silencing as trigger of RNA replicon formation (Mallory et al., 2002, Nature Biotechnol., 20, 622-625), but the system is still impractical, as there is no solution provided for an efficient control of the switch (PTGS suppressor) triggering viral vector replication. However, this system provided for an expression level of the GUS gene reaching 3% of total soluble protein (TSP), which is the best known so far for this type of system, but still no better than a conventional transgene expression system under control of a strong promoter. Another inducible system based on a plant tripartite RNA virus (Mon et al., 2001, Plant J., 27, 79-86), Brome Mosaic Virus (BMV), gave a very low yield of the protein of interest (3-4 μg/g fresh weight), which is comparable with the yields provided by standard transcriptional promoters.
The low expression levels achieved so far with plant expression systems are a major reason why these systems are hardly competitive with other expression systems like bacterial, fungal, or insect cell expression systems. Low expression levels give rise to very high downstream costs for protein isolation and purification in a huge background of plant material. Therefore, costs for downstream processing quickly decrease, as the yield of the protein or product of interest per unit plant biomass increases.
There is presently no large-scale plant transgene expression system the yield and efficiency of which would be sufficiently high to compete on the market with other large-scale expression systems like bacterial, fungal, or insect cell expression systems. Such a plant expression would have to fulfill the following criteria as good as possible:    (i) high yield, including expression of the protein of interest in as many plant tissues as possible and in as many cells of said tissues;    (ii) for preventing a deleterious effect of protein expression on plant growth, expression of the protein or product of interest should be switchable such that expression can be switched on at a desired point in time.    (iii) the switching should be such that expression can be switched on simultaneously or nearly simultaneously in all tissues or cells of a plant and, at the same time, in all plants of a selected group of plants, e.g. in all plants of a selected lot of plants. Typically, the protein or product of interest accumulates in each cell producing said product or protein up to a certain point. During accumulation, however, degradative processes frequently set on that tend to reduce the yield or quality of the protein or product of interest. Therefore, there is an optimal point in time after switching on expression, where the product or protein of interest should be harvested. This optimal point in time should be reached in all tissues or cells of a plant and in all plants of a selected lot at the same time to make the overall process efficient and profitable.
Therefore, it is an object of this invention to provide a transgenic plant, plant part, or plant cell culture for a high-yield plant expression system. It is another object to provide a process of transiently expressing a sequence of interest in a plant, plant part, or plant cell culture. It is another object of the invention to provide an efficient process of expressing one or more sequences of interest in a plant, plant part of plant cell culture, whereby said process can be used efficiently on a large scale. Further, it is an object of the invention to provide a method of controlling the expression of nucleic acid sequence(s) of interest in a plant, plant part, or plant cell culture, which is of improved ecological and biological safety.